Materials and Methods

Introduction and Objective

Conclusions

Results

Rie Matsunaga, Kana Isobe, Megumi Miura, Yuki Kobayashi, Minako Kamihata, Tomoko Maeda, Hiroshi Makino, Kurumi Iwaki, Toshitaka Horiuchi and Masanori Ochi.OCHI YUME CLINIC NAGOYA, Nagoya, Japan.

Sperm DNA fragmentation can reduce the rate of embryo development and also increase the rate ofmiscarriage. Various methods have been developed to select and collect sperm with good motility.However, the DNA fragmentation index (DFI) of the collected sperm differs among these methods. Themicrofluidic sperm selection chamber (ZyMōt™; DxNow) is a selection kit designed to collect low DFIsperm. Although a few reports have suggested a relationship between embryo quality and euploidy rate(assessed by preimplantation testing for aneuploidy after intracytoplasmic sperm injection), there hasbeen little investigation of the efficacy of microfluidic sperm selection (MSS) in IVF. We studied whetherMSS can improve the success rate of embryo development in IVF.

The present study indicates that MSS might enable improvement in the rates of embryo development after IVF of oocytes fromwomen with an advanced maternal age. The use of MSS for sperm selection resulted in the production of a higher frequency of goodquality IVF-embryos with these older patients.

The study was conducted between June and December 2019. Patients were divided into two groupsaccording to the sperm processing method used: Density gradient selection (DGS) (114 patients, 326oocytes), and MSS (113 patients, 356 oocytes) (Table.1, Fig.1).

DGS MSS

patient 114 113

cycle 129 128

oocyte 326 356

age 38.4±4.7 38.3±4.9

Table.1

Experiment 1

We compared embryo development rates between DGS and MSS. Oocytes were divided into fourgroups according to age: under 34 years (A), 35–39 years (B), 40–42 years (C), and over 43 years (D).We studied the rates of blastulation, and available blastocysts (defined as those with Gardner Gradebetter than 4BC); we also compared the rates of available blastocyst cycle, defined as the rate of cyclesthat yielded at least one available blastocyst.

We compared fertilization ratesbetween DGS and MSS. IVF wasperformed at 4×, 6×, and 8×104

sperm/ml respectively, using spermselected by MSS; as a control, 4 x104 sperm in DGS.

Experiment 2

66.6%

44.8%

55.3%

62.4%

0%

10%

20%

30%

40%

50%

60%

70%

80%76.9%

65.2%70.0%

35.3%

71.7%

80.5%

53.6%

62.5%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

A B C D

DGS MSS 53.8%

34.8%36.7%

5.9%

49.1%

53.7%

39.3%

33.3%

0%

10%

20%

30%

40%

50%

60%

A B C D

DGS MSS

83.3%

61.9%64.3%

9.1%

81.3% 80.0%

50.0%53.8%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

A B C D

DGS MSS

• Three layers (45%,68%, 90% percoll)

• Centrifuge 600gfor 15 min

Density gradient

DGS

ZyMōt multi 850

MSS

• Insert semen(850 µl) into the semen chamber

• Add 750µl fertilization medium to upper chamber

• Incubate the device at 37℃ for 30 min

• 6.0% CO2 in air

• Aspirate sperm-containing fluid from the upper retrieval chamber

• Discard the supernatant andresuspend the sperm pelletin modified HTF

• Centrifuge 300g for 5 min

• Discard the supernatant andresuspend the sperm pelletin fertilization medium

Fig.2 Comparison of the fertilization (2PN) rate.*,**P<0.05

The fertilization rates at 4 x, 6 x and 8 x104 sperm/ml were 44.8%, 55.3%, and62.4%, respectively; by comparison, therate in the DGS (control) group was66.6%. Rates of oocyte fertilization usingsperm selected by MSS were significantlylower at 4× and 6×104 sperm/ml than forthe DGS control. However, the rate offertilization improved with increasingsperm concentration in the MSS groupand was comparable to DGS at the8×104 sperm/ml concentration.

Experiment 1 Experiment 2

Fig. 1

***

Blastulation rates did not differ between theDGS and MSS groups in A (76.9% vs.71.7%), B (65.2% vs. 80.5%), C (70.0% vs.53.6%), and D (35.3% vs. 62.5%)(Fig.3).The rates of available blastocysts and theblastocyst cycle rates for MSS werecomparable to DGS in A (49.1% vs. 53.8%and 81.3% vs. 83.3%, respectively), B(53.7% vs. 34.8% and 80.0% vs. 61.9%,respectively), and C (39.3% vs. 36.7% and50.0% vs. 64.3%, respectively). These rateswere significantly higher for MSS than DGSin D (33.3% vs. 5.9% and 53.8% vs. 9.1%,respectively)(Fig.4,5).

Fig.3 Comparison of the blastulation rates per 2PNP>0.05

Fig.4 Comparison of available blastocysts rates per 2PN*P<0.05

*

Fig.5 Comparison of available blastocyst cycle rates per 2PN*P<0.05

*

4 x104 8 x1046 x104 (sperm/ml)

MSSDGS ≤34y 35-39y 40-42y ≥43y

≤34y 35-39y 40-42y ≥43y

≤34y 35-39y 40-42y ≥43y4 x104

217/326

52/116

68/123

73/117

40/52

21/30

15/28

6/17

15/24

38/53

30/46

33/4128/52

11/3011/28

1/17

8/24

26/53

16/46

22/4115/24

10/12

13/16

13/21

12/15

9/14

8/16

1/11

7/13