International Journal of

Molecular Sciences

Article
The State of the Organs of the Female Reproductive System
after a 5-Day “Dry” Immersion
Elena Yu. Gorbacheva 1,2 , Konstantin A. Toniyan 1,2 , Yulia A. Biriukova 2 , Nadezhda A. Lukicheva 1 , Oleg I. Orlov 1 ,
Valery V. Boyarintsev 3 and Irina V. Ogneva 1, *

1 Cell Biophysics Laboratory, State Scientific Center of the Russian Federation Institute of Biomedical Problems
of the Russian Academy of Sciences, 123007 Moscow, Russia
2 Gynecology Department, FGBU KB1 (Volynskaya) UDP RF, 121352 Moscow, Russia
3 Emergency and Extreme Medicine Department, FGBU DPO CGMA UDP RF, 121359 Moscow, Russia
* Correspondence: [email protected]; Tel.: +7-499-195-6398

Abstract: The impact of weightlessness on the female reproductive system remains poorly under-
stood, although deep space exploration is impossible without the development of effective measures
to protect women’s health. The purpose of this work was to study the effect of a 5-day “dry” immer-
sion on the state of the reproductive system of female subjects. On the fourth day of the menstrual
cycle after immersion, we observed an increase in inhibin B of 35% (p < 0.05) and a decrease in
luteinizing hormone of 12% (p < 0.05) and progesterone of 52% (p < 0.05) compared with the same
day before immersion. The size of the uterus and the thickness of the endometrium did not change.
On the ninth day of the menstrual cycle after immersion, the average diameters of the antral follicles
and the dominant follicle were, respectively, 14% and 22% (p < 0.05) higher than before. The duration
of the menstrual cycle did not change. The obtained results may indicate that the stay in the 5-day
“dry” immersion, on the one hand, can stimulate the growth of the dominant follicle, but, on the
other hand, can cause functional insufficiency of the corpus lutea.

Keywords: microgravity; ovary; endometrium; uterus; anti-Mullerian hormone; inhibin B; follicle-

stimulating hormone; luteinizing hormone; progesterone
Citation: Gorbacheva, E.Y.; Toniyan,
K.A.; Biriukova, Y.A.; Lukicheva,
N.A.; Orlov, O.I.; Boyarintsev, V.V.;
Ogneva, I.V. The State of the Organs
of the Female Reproductive System 1. Introduction
after a 5-Day “Dry” Immersion. Int. J. Space exploration opens up new possibilities for the development of mankind. At the
Mol. Sci. 2023, 24, 4160. https:// same time, maintaining the health of the various systems in the body during space flight
doi.org/10.3390/ijms24044160 and the subsequent adaptation to the Earth’s gravity continues to be an important task. In
Academic Editors: Khaled Kamal this context, little attention has been paid to the health of the reproductive system since a
and John Lawler change in its condition does not pose an immediate critical threat to life.
Although space exploration has been historically dominated by men, more than
Received: 23 January 2023 65 women have already made at least one space flight, and for them, the state of the
Revised: 15 February 2023
reproductive system is of fundamental importance from at least two points of view. On
Accepted: 18 February 2023
the one hand, healthy aging in the female body depends on hormonal balance, which
Published: 19 February 2023
supports the health of the reproductive system. On the other hand, the average age
of women on their first flight is 38 years [1], which, even under Earth conditions, is
considered a late reproductive age. Despite the fact that the average age for the birth of
Copyright: © 2023 by the authors.
the first child in women preparing for space flight is about 35 years [1], many of them
Licensee MDPI, Basel, Switzerland. postpone starting a family until they have made several space flights [2]. In addition, under
This article is an open access article the conditions of space flight, women often suppress the menstrual cycle by taking oral
distributed under the terms and contraceptives [3]. After a space flight, some women are able to successfully conceive,
conditions of the Creative Commons even with the use of assisted reproductive technologies [4]. However, the proportion of
Attribution (CC BY) license (https:// unsuccessful reproductive efforts is quite high but the data are insufficient to assess the
creativecommons.org/licenses/by/ impact of space flight on reproductive function [2,4], as well as the state of the female
4.0/). reproductive system after space flight [1].

Int. J. Mol. Sci. 2023, 24, 4160. https://doi.org/10.3390/ijms24044160 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2023, 24, 4160 2 of 12

Research under space flight conditions is a priority; however, obtaining reliable results
is difficult due to a small sample size. Therefore, various ground-based models are used,
primarily the 6◦ head-down-tilt bed-rest model. This model has been used to study the
state of the female reproductive system [5] but the data are scarce and contradictory. In
one study, after a 17-day 6◦ head-down-tilt bed-rest test, there was no change in the length
of the menstrual cycle [6]. In another study, the authors noted luteal phase deficiency
in 3 out of 12 subjects by measuring progesterone levels [7]. However, despite the fact
that female subjects participated in complex bed-rest experiments [8–11], nevertheless, the
reproductive system was not the main priority.
Another generally accepted ground-based model is “dry” immersion [12–14]. How-
ever, similar to the bed-rest model, studies during “dry” immersion are most often aimed
at the musculoskeletal, nervous, and cardiovascular systems [15–18]. Moreover, the first
“dry” immersion study with women as the subjects that lasted 3 days was conducted in
2020 [19].
In this paper, we present the results of a study of the effect of 5-day “dry” immersion
on the female reproductive system. The beginning of the exposure in the immersion bath
was tied to the 10th day of the menstrual cycle and, accordingly, the exposure coincided
with the late follicular phase and ovulation. We evaluated the content of the hormones
involved in the maturation of follicles and the formation of corpus lutea and performed an
ultrasound examination aimed at determining the structural parameters of the organs of
the female reproductive system.

2. Results
2.1. The Levels of Hormones Involved in the Functioning of the Reproductive Tissue
Anti-Mullerian hormone is a marker of ovarian reserve, in particular, the pool of
primary follicles. The median value of anti-Mullerian hormone (AMH) concentrations in
the blood serum before and after immersion did not differ significantly (Figure 1A). The
individual data showed that the concentration of AMH before and after immersion did
not go beyond the reference values, except for two subjects. In these subjects, the AMH
concentrations before immersion were above the upper limit of normal; after immersion,
these levels decreased but did not reach the normal level.
Like AMH, inhibin B is a marker of the ovarian reserve but it marks the pool of
secondary, pre-, and antral follicles. After immersion, the concentration of inhibin B in-
creased; the median value after immersion was 35% higher (p < 0.05) than before immersion
(Figure 1B). The individual data showed that the concentration of inhibin B before and
after immersion remained within the normal range in all subjects.
The concentration of follicle-stimulating hormone (FSH), on average, did not change
after immersion; in all subjects, the values before and after immersion were within the
normal range (Figure 1C).
In 10 out of 12 subjects, the concentration of luteinizing hormone (LH) decreased after
immersion; in 1 it did not change and in 1 it increased (Figure 1D). The median value
after immersion was 12% lower (p < 0.05) than before immersion. In one subject, the LH
concentration exceeded the reference level before immersion; however, after immersion, it
decreased and corresponded to the norm.
The concentration of progesterone (PG) generally decreased after immersion among
the subjects and in 8 of 12 subjects, it fell below the lower limit of the norm (Figure 1E).
However, in five subjects, even before immersion, the PG concentration was below the norm.
The median value after immersion was lower than before immersion by 52% (p < 0.05).
 Int. J.J.Mol.
Int. Mol. Sci.
Sci.2023, 24,x4160
2023,24, FOR PEER REVIEW 3 3ofof 12
12

Figure 1.
Figure The concentration
1. The concentrationof ofhormones
hormonesbefore
beforeand after
and a 5-day
after “dry”
a 5-day immersion.
“dry” immersion. (A) anti-Mullerian
(A) anti-Mul-
hormone (AMH), (B) inhibin B, (C) follicle-stimulating hormone (FSH), (D)
lerian hormone (AMH), (B) inhibin B, (C) follicle-stimulating hormone (FSH), (D) luteinizing luteinizing hormone
hor-
(LH), (LH),
mone (E) progesterone (PG). (PG).
(E) progesterone The individual data are
The individual presented
data on theon
are presented left
theside
leftofside
each
of panel (1–12
each panel
(1–12 are
are the the conditional
conditional numbers
numbers of the of the subjects).
subjects). The circles
The black black circles indicate
indicate the values
the values before im-
before immersion,
mersion, the red
the red circles circles indicate
indicate the valuestheafter
values after immersion,
immersion, and the and the direction
direction of change of change is indicated
is indicated in blue
in blue (↑—increase,
(↑—increase, ↓—decrease,
↓—decrease, ~—unchanged).
~—unchanged). Data
Data are are presented
presented on theside
on the right right
of side
eachofpanel
eachfor
panel
the
for the median ± range group. Before—value before immersion. After—value after immersion. *p <
median ± range group. Before—value before immersion. After—value after immersion. * p < 0.05 in
0.05 in comparison to “before”. The green dotted lines indicate the upper or lower limit of the norm,
comparison to “before”. The green dotted lines indicate the upper or lower limit of the norm, if, for
if, for some subjects, the values were outside the reference range. The reference concentration is
some subjects,
shown the values
on a vertical were
scale for outside
each the reference range. The reference concentration is shown on
hormone.
a vertical scale for each hormone.
 Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 4 of 12

Int. J. Mol. Sci. 2023, 24, 4160 4 of 12

Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW
2.2. The Data of the Ultrasound Examination of the Organs of the Reproductive System 4 of 12
According to ultrasound data, the size of the uterus before and after immersion did
not differ on both the fourth and ninth days of the menstrual cycle (dmc) (Figure 2A).
2.2. The Data ofthickness
Endometrial the Ultrasound
also didExamination of the and
not change before Organs
afterofimmersion
the Reproductive System
on the correspond-
2.2. The Data of the Ultrasound Examination of the Organs of the Reproductive System
ing According
days of the menstrual cycle (Figure 2B).
to ultrasound data, the size of the uterus before and after immersion
According to ultrasound data, the size of the uterus before and after immersion did
did not differ on both the fourth and ninth days of the menstrual cycle (dmc) (Figure 2A).
not differ on both the fourth and ninth
12
days of the menstrual cycle (dmc) (Figure 2A).

thickness, mm
75 4 dmc Endometrial
Endometrialthickness
thicknessalso
9 dmc
alsodid
did not
not change beforeand
change before andafter
afterimmersion
immersion 9 on
on dmcthe corresponding
the correspond-
days of the menstrual cycle (Figure 2B).
mm size, mm

60
ing days of the menstrual cycle (Figure9 2B).
4 dmc

45 12

thickness, mm
75 4 dmc 9 dmc 6 9 dmc
Uterus size, Uterus

length length

Endometrium
width width
30
60
depth depth 39 4 dmc

15
45
length length 06

Endometrium
width width
before after before after
30
A depth depth B 3
15
Figure 2. The size of the uterus (A) and 0the thickness of the endometrium (B) on the 4th and 9th
days of the menstrual cycle (dmc) before andbefore after
after a 5-day before
“dry” immersion. after
A B
The average volume of the ovaries of the subjects on the fourth day of the menstrual
Figure
Figure
cycle 2. 2.The
did Thesize
not sizeofofthe
differ theuterus
uterus
before (A)
and and
andthe
(A)after thethickness
the thickness
5-day of
of the
“dry” the endometrium
endometrium
immersion. (B) on
(B)
However, on the
the 4th
on4th and
theand 9th days
9th
ninth
ofday
the menstrual
days of the cycle
menstrual (dmc)
cycle before
(dmc) and
before after
and a 5-day
after a “dry”
5-day “dry”immersion.
immersion.
of the menstrual cycle, the ovarian volume after immersion decreased, on average,
for the group; the median value after immersion was 22% lower (p < 0.05) than before
The
Theaverage
immersion average
(Figure volume
volume of the
3A,B). of theovaries
ovariesofofthe the subjects
subjects on on
thethe fourth
fourth day day ofmenstrual
of the the menstrual
cycle did
cycleThe not
didaveragediffer
not differ before and
before of
diameter and after
theafter the 5-day
thefollicles
antral “dry”
5-day “dry” immersion.
on theimmersion. However,
fourth dayHowever, on
on the
of the menstrual theninth
ninth day
cycle
day
the of
ofbefore the after
menstrual
and menstrual
cycle, cycle,
immersion the not
the ovarian
did ovarian
differvolume
volume afterafter immersion
immersion
significantly, thedecreased,
but on decreased,
ninth day onofon average,
average,
the cyclefor the
for the
group;
after the group;
median
immersion, the median
it value value
after
increased after
byimmersion
14% (p <immersion
wasas22%
0.05), was
did 22%
lower
the (plower
diameter of(pthe
< 0.05) <than
0.05)before
than follicle
dominant before
immersion
immersion
(Figure
by (p < (Figure
22%3A,B). 0.05). 3A,B).
The average diameter of the antral follicles on the fourth day of the menstrual cycle
before and after immersion did not differ significantly, but on the ninth day of the cycle
after immersion, it increased by 14% (p < 0.05), as did the diameter of the dominant follicle
by 22% (p < 0.05).

Figure3.3.Ovarian
Figure Ovarian volume andfollicle
volume and follicle diameter
diameter before
before andand
afterafter a 5-day
a 5-day “dry”“dry” immersion
immersion on dayson
4 days
and 99 of
4 and of the
the menstrual
menstrualcycle.
cycle.(A)(A)
Average ovarian
Average volume
ovarian for the
volume for group. (B) An
the group. (B)example of an of an
An example
ultrasound picture: right ovary, 9th day of the menstrual cycle of the same subject before and after
immersion—ovary volume decreased from 14 mL to 8.4 mL. Yellow dotted lines show the ovary sizes
Figure 3. Ovarian volume and follicle diameter before and after a 5-day “dry” immersion on days 4
in and
the 9three dimensions,
of the which(A)
menstrual cycle. areAverage
indicated by numbers
ovarian volume for1, 2,
the3 group.
in the image
(B) An (C) Average
example follicle
of an
diameter for the group. (D) An example of an ultrasound picture: 9th day of the menstrual cycle of
the same subject before and after immersion—dominant follicle diameter increased from 12 mm to
18 mm. Yellow dotted lines mark the size of the dominant follicle (a 2x zoom is used in the “before”
picture). * p < 0.05 in comparison to “before” of the same day of the menstrual cycle (dmc).
Int. J. Mol. Sci. 2023, 24, 4160 5 of 12

The average diameter of the antral follicles on the fourth day of the menstrual cycle
before and after immersion did not differ significantly, but on the ninth day of the cycle
after immersion, it increased by 14% (p < 0.05), as did the diameter of the dominant follicle
by 22% (p < 0.05).

3. Discussion
To date, there are practically no data on the impact of space flight or simulated
weightlessness on the female reproductive system. The results of experiments on rodents
are also scarce and incomparable due to the use of different species. Thus, in female
Sprague–Dawley rats with a normal cycle, antiorthostatic suspension led to an elongation
of the estrous cycle, which was characterized by more time spent in the diestrus stage and
less time spent in the oestrus stage. In addition, antiorthostatic suspension was associated
with a decrease in the serum estradiol concentration, the levels of LH and FSH in the serum
and pituitary gland did not change, the level of progesterone in the serum did not change,
and the weight of the ovaries and uterus did not change but the weight of the pituitary
gland decreased [20]. However, Holets H.M. et al. showed that after space flight (shuttle
missions STS-131, 133, 135), female mice experienced cycling arrest, loss of corpus lutea,
and decreased estrogen receptor expression in the uterus [1]. In vitro experiments with
mouse ovarian tissues showed that under conditions of simulated microgravity for 48 and
96 h, there was a decrease in the number of secondary follicles per unit area, there was no
proliferation of granulosa cells, and there was no secretion of growth differentiation factor-9
(GDF9) by oocytes [21]. Rodent studies allow us to study the subtle mechanisms of the
migration of primary germ cells and the formation of a pool of primordial follicles, as well
as their recruitment to growth; however, in relation to humans, one should take into account
the different ovulatory cycle (menstrual vs. estrus) and species-specific regulatory factors.
In humans, as in other mammals, the formation of follicles begins during embryo-
genesis. Primordial germ cells simultaneously divide and migrate to the gonadal ridge
where their cell cycle stops at the stage of the prophase of the first meiotic division, and a
primordial follicle is formed with a diameter of less than 0.1 mm [22,23]. The maximum
number of these follicles is observed around the 20th week of the development of the female
fetus (more than 7 million), and then their death begins, which continues throughout the
life of the woman [23,24]. These follicles form the ovarian reserve and a cohort of growing
follicles are recruited from their pool.
The activation of the growth of the primordial follicle leads to the surrounding squa-
mous epithelial cells (pre-granulosa cells) becoming cubic granulosa cells and the formation
of the primary follicle. The further growth of the oocyte, proliferation of granulosa cells
up to 2 or more layers, and formation of the basement membrane and theca cells around
it lead to the formation of a secondary follicle. The interaction between the oocyte and
its surrounding cells determines the growth of the follicle [25]. The oocyte produces the
GDF9 and BMP15 factors that stimulate the growth of granulosa cells [26]. Granulosa cells
produce AMH, which, in turn, blocks the recruitment of new primordial follicles, activins
that stimulate theca cell growth, and inhibins that block FSH secretion. Theca cells also
produce growth factors TGFβ and LH receptors; however, activins block steroidogenesis in
theca cells at this stage [25,27]. This preantral follicle is about 0.2 mm in size [28] and does
not depend on gonadotropins.
The follicle continues to grow and a fluid-filled cavity (antrum) begins to form within
it, dividing the granulosa cells into mural granulosa and cumulus cells, directly surround-
ing the oocyte. The small antral follicle is 0.2–0.4 mm in size [28] and at this stage, FSH
receptors appear on granulosa cells. Follicle growth begins to depend on the hypothalamo–
pituitary–gonadal axis [23,29]. From the pool of antral follicles, which are 2–5 mm in
size [23,30], under the action of FSH and LH, the dominant follicle will mature and ovulate.
Theca cells with receptors for LH under their control begin the synthesis of androstene-
dione from which estradiol is synthesized in granulosa cells under the control of FSH.
Increasing concentrations of inhibin B and estradiol suppress FSH secretion and stimulate
Int. J. Mol. Sci. 2023, 24, 4160 6 of 12

the formation of LH receptors on granulosa cells [25,31]. The peak release of LH leads to
ovulation. The granulosa and theca cells remaining after ovulation are luteinized under the
action of LH and begin to produce progesterone [25,27].
The cycle of follicle growth from primordial to ovulatory takes about 175 days [32],
i.e., about 6–7 menstrual cycles. There is no single point of view on the growth dynamics
of the recruited follicles [23]; however, taking into account the duration of the full cycle,
we can say that the antral follicles we assessed after immersion were in the growth phase
during immersion.
The results obtained indicate that the AMH content did not differ before and after
immersion (Figure 1A), which may indicate that the recruitment of the pool of dormant
primordial follicles did not change. Notably, the possible effect of the 5-day immersion
on the pool of primordial follicles can only be assessed after about six months, due to the
duration of the follicle growth cycle (see above).
However, the content of inhibin B significantly increased after immersion (Figure 1B).
Inhibin B is synthesized by the granulosa cells of the growing follicles so we can assume
an increase in the number of cells and/or an increase in the expression of inhibin B. The
latter seems more likely since the diameter of the follicle, according to ultrasound, did not
change on this (fourth) day of the cycle before and after immersion (Figure 3C). Before the
small antral follicle stage, both AMH and inhibin B are synthesized by the granulosa cells
of the growing follicle [27,33] but we did not observe changes in the AMH content. The
expression of inhibin B continues at the stage of a large antral follicle so it can be assumed
that changes in granulosa cells take place at this stage.
The peak value of inhibin B content is observed at a follicle diameter of about
11 mm [31] and, like estradiol, inhibin B suppresses FSH synthesis in the mid-follicular
phase [31]. We analyzed the FSH content on the fourth day of the cycle and found no
change after immersion (Figure 1C). However, on the ninth day of the cycle after immersion,
the diameter of the antral follicles was significantly higher, as well as that of the dominant
follicle (Figure 3C). Moreover, the diameter of the dominant follicle was, on average, more
than 15 mm, which may indirectly indicate a shortening of the follicular phase and ap-
proaching ovulation. However, it should be noted that the subjects did not notice a change
in the length of the menstrual cycle during which they were in dry immersion conditions
from the usual duration.
However, the level of LH in the blood on the fourth day of the cycle was significantly
lower after immersion than before (Figure 1D). The decrease in LH may be associated with
a decrease in its secretion by the pituitary gland but we did not find similar data in humans
under microgravity conditions. Notably, in rodents after antiorthostatic suspension, a
decrease in the mass of the pituitary gland was observed, although without a change in
the content of gonadotropins [20]. On the other hand, a decrease in LH in the blood may
be due to an increase in its utilization in the ovarian tissue. Inhibin B inhibits the action
of activin, which suppresses the synthesis of LH receptors [25]. In other words, inhibin
B has a stimulating effect on the expression of the LH receptor by theca cells, which, in
turn, can lead to an increase in the uptake of LH from the blood and a decrease in its
concentration there.
Luteinizing hormone leads to the formation of corpus lutea and controls its synthesis
of progesterone. Accordingly, the decrease in LH could explain the decrease in blood
progesterone observed after immersion (Figure 1E). Moreover, after bed rest, a decrease
in progesterone was also noted in women [7] and the absence of corpus lutea formation in
mice after space flight [1]. However, on the fourth day of the menstrual cycle, when we
conducted the study, the corpus lutea of the previous cycle regressed and a new one will
form only after ovulation.
However, progesterone synthesis begins even before ovulation LH surge by granulosa
cells through the prostaglandin E2/EP2 receptor pathway stimulated by GDF9 [34–36].
If we rely on the above assumption about the shortening of the follicular phase and
the shift of the ovulation time to the left, then it can be assumed that the decrease in
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 7 of 12

However, progesterone synthesis begins even before ovulation LH surge by granu-

Int. J. Mol. Sci. 2023, 24, 4160 7 of 12
losa cells through the prostaglandin E2/EP2 receptor pathway stimulated by GDF9 [34–
36]. If we rely on the above assumption about the shortening of the follicular phase and
the shift of the ovulation time to the left, then it can be assumed that the decrease in pro-
progesterone
gesterone content
content is associated
is associated with awith a possible
possible decreasedecrease
in GDF9 in synthesis
GDF9 synthesis under
under condi-
conditions of simulated microgravity, which was shown in in vitro
tions of simulated microgravity, which was shown in in vitro experiments with mouseexperiments with mouse
tissues[21].
tissues [21]. Although
Althoughititshould
shouldbe benoted
notedthat
thatprogesterone,
progesterone,in in minimal
minimalamounts,
amounts,isis also
also
synthesized in somatic tissues, which we have not studied, which makes
synthesized in somatic tissues, which we have not studied, which makes our assumptions our assumptions
ratherspeculative.
rather speculative.
Summarizing
Summarizing the theresults
resultsobtained
obtained andandthethe above
above assumptions,
assumptions, we propose
we can can propose
a pos-a
possible
sible sequence
sequence of events
of events (Figure(Figure
4) that 4) that
lead lead
to an to an increase
increase in the of
in the diameter diameter of the
the dominant
dominant follicle, an increase in the concentration of inhibin B in the blood,
follicle, an increase in the concentration of inhibin B in the blood, and a decrease in the and a decrease
in the concentration
concentration of LH
of LH and and progesterone.
progesterone.

BEFORE AFTER

pituitary pituitary
gland gland
FSH FSH
LH LH
1
blood vessel blood vessel
1- inhibin B
FSH FSH inhibin B Dominant follicle
FSH
d = 15 mm
Dominant follicle inhibin B LH LH
d = 12.3 mm estradiol
5-day “dry”
immersion

estradiol

oocyte
2
oocyte

LH LH
androgens 2 - LH androgens

progesterone 3 - progesterone progesterone

cholesterol 3
cholesterol

Figure
Figure 4.4. Possible
Possibleschemeschemeof ofevents
eventsleading
leadingto toaachange
changein in the
the concentration
concentrationof ofhormones
hormonesand andthe
the
diameter
diameterof ofthethedominant
dominantfollicle
follicleafter
afteraa 5-day
5-day “dry”
“dry” immersion.
immersion.Changes
Changesin inhormone
hormonecontent
contentareare
labeled
labeledas asfollows:
follows:↑—increase,
↑—increase, ↓—decrease,
↓—decrease, ~—unchanged.
~—unchanged. (1) As
(1) aAsresult of staying
a result in “dry”
of staying im-
in “dry”
mersion
immersion from 10 to1015todays
from during
15 days the previous
during menstrual
the previous cycle,cycle,
menstrual an increase in the in
an increase number of gran-
the number of
ulosa cells of the growing antral follicles occurs. This leads to an increase in the growth of the dom-
granulosa cells of the growing antral follicles occurs. This leads to an increase in the growth of the
inant follicle in the next cycle and an acceleration of its maturation. Thus, the diameter of the dom-
dominant follicle in the next cycle and an acceleration of its maturation. Thus, the diameter of the
inant follicle increases, and, due to the increase in the number of granulosa cells, the production of
dominant
inhibin follicle increases,
B increases (markedand, withdue to the
a small increase
green in the
circle), number
as well of concentration
as its granulosa cells, inthe
theproduction
blood. (2)
of inhibin B increases (marked with a small green circle), as well as
Accelerated maturation of the follicle leads to an earlier appearance of LH receptors onits concentration in thegranulosa
blood. (2)
Accelerated
cells maturationto
and, consequently, oftheir
the follicle
greaterleads to an earlier
utilization appearance ofAccordingly,
in the bloodstream. LH receptorsthe onconcentra-
granulosa
tion
cellsofand,
LHconsequently,
(marked withtoa their smallgreater
light-blue circle) in
utilization in the blood falls. (3)Accordingly,
bloodstream. A decrease in thethe concentra-
concentration
tion of LH in the blood leads to its deficiency for receptors located on the theca
of LH (marked with a small light-blue circle) in the blood falls. (3) A decrease in the concentration cells and, conse-
quently, to a decrease in the synthesis of progesterone (marked with a small
of LH in the blood leads to its deficiency for receptors located on the theca cells and, consequently, yellow circle) and a
decrease in its concentration in the blood. The arrows indicate the migration
to a decrease in the synthesis of progesterone (marked with a small yellow circle) and a decrease of hormones, the blue
in
dashed line indicates the fluxes that have changed after immersion.
its concentration in the blood. The arrows indicate the migration of hormones, the blue dashed line
indicates the fluxes that have changed after immersion.
4. Materials and Methods
4. Materials
4.1. andDesign
Experimental Methods
4.1. Experimental Design
Sixteen healthy women were recruited for the experiment. For the study of the
reproductive system described in this article, 12 out of 16 subjects were selected and
4 subjects were excluded because they were taking oral contraceptives. Of the 12 subjects,
 the waterproof fabric) up to neck level in a supine position. The area of the fabric’s surface
considerably exceeded that of the water’s surface. The high elasticity properties of the
fabric artificially created conditions similar to zero gravity via floatation. The folds of the
waterproof fabric allowed the person’s body to be freely enveloped on all sides and avoid
any contact of the body with water. Subjects lay in the immersion bath without any phys-
Int. J. Mol. Sci. 2023, 24, 4160 ical activities. The average time spend outside the immersion bath (for hygiene proce- 8 of 12
dures and certain experimental examinations) did not exceed 30 min per day. The crew,
consisting of a doctor, an assistant, and a technician, provided 24 h monitoring of partici-
pants’ health
6 received and the working
a lactoferrin conditions
preparation once a ofdaythe technical
from days 1equipment
to 29 of the[37].
experiment and the
remaining 6 received a placebo. In our study data, there wasimmersion
The beginning of exposure under the conditions of “dry” (DI) between
no difference in 7 subjects
the
was on the 10th
parameters day ofwho
of subjects the received
menstrual thecycle
drug(dmc), in 3 who
and those subjects at 12 admc,
received in 1 Therefore,
placebo. subject at
13
wedmc, in 1 the
consider subject at 15group
general dmc.to All
besubjects at 4 dmcsubjects.
the 12 selected underwent blood sampling (strictly
on anThe
emptyagestomach) for estimating
of the subjects ranged the fromlevels
22.7oftohormones
40.8 years and an the
and ultrasound
averageexamina-
age was
tion
27.8 for estimating
years the antral
(Figure 5A). follicles.ofAtthe
The duration 9 dmc, only an
menstrual ultrasound
cycle examination
during the experimentwas per-
ranged
formed for estimating the dominant follicle. The study was carried out before
from 26 to 32 days, with an average of 29 days, and the subjects did not notice a change in and after
exposure
the lengths toof
dry immersion
their menstrualconditions (Figure
cycles (Figure 5C).
5B).

dmc
45 Duration of menstrual cycle, days 33 29
1
4 Blood sampling, USI
40

Menstrual cycle
31 9 USI
10
Age, years

DI
35 15
29
30
27 29
25 1
4 Blood sampling, USI

20 25 9 USI

time line
A B C

Figure 5. Characteristics
Characteristics of
of the
thegroup
groupofofsubjects.
subjects.(A)
(A)Average
Average ageage
of of
thethe subjects.
subjects. (B)(B) Duration
Duration of
of the
the menstrual
menstrual cycle.
cycle. (C) (C) Cyclogram
Cyclogram of the
of the study
study of the
of the state
state of the
of the reproductive
reproductive system.
system. dmc—day
dmc—day of
menstrual cycle, DI—“dry” immersion, USI—ultrasound imaging. The cyclogram is given for the
average duration of the menstrual cycle in the group of subjects (29 days) and the start of exposure at
10 dmc.

Exposure under the conditions of “dry” immersion for 5 days was carried out exactly
as described earlier for a 3-day immersion [37]. It is known that most physiological systems
under the conditions of a “dry” immersion respond after 3 days, and the maximum acute
changes are observed after 5–7 days [14]. Since the ongoing immersion study is a complex
experiment aimed at studying all the physiological systems of the body, a duration of
5 days was chosen. The exposure was conducted at the “dry” immersion facility of the
Institute of Biomedical Problems, Russian Academy of Science [14,19]. Subjects, wearing a
shirt and trunks, were placed on waterproof fabric and immersed in a deep bath (the water
temperature in the bath was maintained at 32.5 ± 2 ◦ C to avoid body cooling through the
waterproof fabric) up to neck level in a supine position. The area of the fabric’s surface
considerably exceeded that of the water’s surface. The high elasticity properties of the
fabric artificially created conditions similar to zero gravity via floatation. The folds of the
waterproof fabric allowed the person’s body to be freely enveloped on all sides and avoid
any contact of the body with water. Subjects lay in the immersion bath without any physical
activities. The average time spend outside the immersion bath (for hygiene procedures and
certain experimental examinations) did not exceed 30 min per day. The crew, consisting of
a doctor, an assistant, and a technician, provided 24 h monitoring of participants’ health
and the working conditions of the technical equipment [37].
The beginning of exposure under the conditions of “dry” immersion (DI) in 7 subjects
was on the 10th day of the menstrual cycle (dmc), in 3 subjects at 12 dmc, in 1 subject at
13 dmc, in 1 subject at 15 dmc. All subjects at 4 dmc underwent blood sampling (strictly on
an empty stomach) for estimating the levels of hormones and an ultrasound examination
for estimating the antral follicles. At 9 dmc, only an ultrasound examination was performed
Int. J. Mol. Sci. 2023, 24, 4160 9 of 12

for estimating the dominant follicle. The study was carried out before and after exposure
to dry immersion conditions (Figure 5C).
For our investigation of the reproductive system, all subjects were treated at Gyne-
cology Department Clinical Hospital # 1 (Volynskaya, Moscow, Russia). All subjects were
in a stable clinical condition with no clinical, microbiological, or laboratory evidence of
infection, encephalopathy, renal failure, or comorbidities including heart failure, pulmonary
disease, malignancy, or diabetes mellitus. Written informed consent was obtained from
each subject prior to participation in the study. The study design and procedures were
approved by the Biomedicine Ethics Committee of the Institute of Biomedical Problems,
Russian Academy of Sciences (Physiology Section of the Russian Bioethics Committee,
Russian Federation National Commission for UNESCO, Permit #615/MSK/06/06/22) and
conformed to the Declaration of Helsinki.

4.2. Blood Sampling and Measurements of Hormones

Blood samples were collected at around 8.30 am for all subjects at 4 dmc before
and after DI. Serum was separated by centrifugation and measurements were made im-
mediately without freezing. Follicle-stimulating hormone (FSH), luteinizing hormone
(LH), anti-Mullerian hormone (AMH), and progesterone (PG) were determined by elec-
trochemiluminescence immunoassay (Elecsys® reagents and Cobas e411 analyzer, Roche
Diagnostics Ltd., Rotkreuz, Switzerland). Inhibin B was measured using an enzyme-linked
immunosorbent kit (#A81303, Beckman Coulter, Inc., Brea, CA, USA) by an automatic
analyzer (Personal LAB, Adaltis S.r.l., Rome, Italy).

4.3. Ultrasound Examination

Ultrasound examinations of the organs of the reproductive system were performed at
4 dmc and 9 dmc before and after DI using an EPIQ7 ultrasound system with a broadband
curved array transducer with PureWave crystal technology C10-3v (Philips, Amsterdam,
Netherlands). Imagery for determining the size of the uterus and ovaries, the diameter of
the follicles, as well as counting their number, was performed using the nSIGHT integrated
imaging system (Philips, Amsterdam, Netherlands).

4.4. Statistical Analysis

We used non-parametric statistics due to the small number of subjects, high variability
of the levels of the baseline parameters, and non-normal distribution. To compare “before”
and “after”, Wilcoxon tests were used for post hoc comparisons with statistical significance
at the p < 0.05 level. Data were presented as median ± range.

5. Conclusions
Summarizing the above, we can conclude that even a 5-day stay under the conditions
of “dry” immersion in the late follicular phase/ovulation affects the state of the female
reproductive system. The ovarian reserve remains intact since its main marker—anti-
Mullerian hormone—does not change. The growth of antral follicles is likely stimulated—
their diameter and that of the dominant follicle increase, as well as the concentration of
inhibin B. At the same time, the concentrations of luteinizing hormone and progesterone
on the fourth day of the cycle after immersion were significantly reduced compared to the
corresponding values before immersion. Very cautiously, we can assume that this was due
to a shortening of the follicular phase and a shift of ovulation to the left, although other
explanations are possible, which would require further research.

6. Limitations of the Study

There are several limitations of this study:
- the small number of subjects and high variability of the baseline parameters;
Int. J. Mol. Sci. 2023, 24, 4160 10 of 12

- the phase of the menstrual cycle was determined by the date of the beginning of the
last menstrual period but, due to the different reproductive histories, ages, etc., it
could have shifted the longevity of the phases;
- due to technical limitations during the organization of the experiment, the different
days of the menstrual cycle at the beginning of exposure in the immersion bath could
have caused the variability of some of the effects;
- due to the same reasons mentioned above, our examinations were possible only on the
fourth and ninth days of the menstrual cycle. The optimal dmc for estimating hormone
levels is 2–3 dmc, for counting antral follicles it is 5–6 dmc, and for estimating the
dominant follicle it is 11–12 dmc. In addition, it would be better to expand the range
of detectable hormones involved in the functioning of the reproductive tissue, as well
as the points of measurement. The lack of subject follow-up over future menstrual
cycles and, as the result, the lack of data on the dynamics of the measured parameters
is one of the most significant limitations.

Author Contributions: Conceptualization, E.Y.G., K.A.T., O.I.O., V.V.B. and I.V.O.; methodology,
E.Y.G., K.A.T., Y.A.B., N.A.L. and I.V.O.; software, E.Y.G. and I.V.O.; validation, E.Y.G., K.A.T., Y.A.B.,
N.A.L., V.V.B. and I.V.O.; formal analysis, E.Y.G., Y.A.B. and I.V.O.; investigation, E.Y.G., K.A.T.,
Y.A.B. and I.V.O.; resources, N.A.L., O.I.O., V.V.B. and I.V.O.; data curation, E.Y.G., K.A.T., Y.A.B. and
I.V.O.; writing—original draft preparation, E.Y.G. and I.V.O.; writing—review and editing, E.Y.G.,
K.A.T., Y.A.B., N.A.L., V.V.B. and I.V.O.; visualization, E.Y.G. and I.V.O.; supervision, I.V.O.; project
administration, N.A.L., O.I.O., V.V.B. and I.V.O.; funding acquisition, N.A.L., O.I.O., V.V.B. and I.V.O.
All authors have read and agreed to the published version of the manuscript.
Funding: This work was financially supported by the program for fundamental research SSC RF—
IBMP RAS 65.4 and UNU “Transgenbank”.
Institutional Review Board Statement: The study design and procedures were approved by the
Biomedicine Ethics Committee of the Institute of Biomedical Problems, Russian Academy of Sciences
(Physiology Section of the Russian Bioethics Committee, Russian Federation National Commission
for UNESCO, Permit #615/MSK/06/06/22) and conformed to the Declaration of Helsinki.
Informed Consent Statement: Written informed consent has been obtained from each subject prior
to participation in the study and the publishing of this paper. All subjects were in a stable clinical
condition with no clinical, microbiological, or laboratory evidence of infection, encephalopathy, renal
failure, or comorbidities, including heart failure or pulmonary disease, malignancy, or diabetes
mellitus.
Data Availability Statement: All data generated or analyzed during this study are included in
this article.
Acknowledgments: The authors are very grateful to all who organized the “dry” immersion and its
participants.
Conflicts of Interest: The authors declare no conflict of interest.

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